In this study, we show a correlation between synthesis of aberrant proteins and their oxidative modification. The level of aberrant proteins was elevated in Escherichia coli cultures by decreasing transcriptional or translational fidelity using specific mutations or drugs. Protein carbonylation, an oxidative modification, increased in parallel to the induction of the heat shock chaperone GroEL. As the protein turnover rates and level of intracellular oxidative stress remained unchanged, it appears that carbonylation results from the increased susceptibility of the misfolded proteins. These studies show that the cellular protein oxidation is not limited only by available reactive oxygen species, but by the levels of aberrant proteins. Thus, protein oxidation seen in aging cells may be the consequence also of reduced transcriptional/translational fidelity, and protein structures appear to have evolved to minimize oxidative damage. In addition, we discuss the possibility that carbonylation, being an unrepairable protein modification, may serve as a tagging system to shunt misfolded proteins between pathways of refolding by chaperones or the proteolytic apparatus.

Aging, or senescence, is the progressive deterioration of every bodily function over time. A fundamental question that applies to all life forms, including growth-arrested bacteria, is why growing older by necessity causes organisms to grow more fragile. In this work, we demonstrate that the levels of oxidized proteins is correlated to the age of a stationary-phase Escherichia coli culture; both disulfide bridge formation of a cytoplasmic leader-less alkaline phosphatase and protein carbonyl levels increase during stasis. The stasis-induced increase in protein oxidation is enhanced in cells lacking the global regulators OxyR and s . Some proteins were found to be specifically susceptible to stasis-induced oxidation; notably several TCA cycle enzymes, glutamine synthetase, glutamate synthase, pyruvate kinase, DnaK, and H-NS. Evidence that oxidation of target proteins during stasis serves as the signal for stationary-phase, developmental, induction of the heat shock regulon is presented by demonstrating that this induction is mitigated by overproducing the superoxide dismutase SodA. In addition, cells lacking cytoplasmic superoxide dismutase activity exhibit superinduction of heat shock proteins. The possibility that oxidative sensitivity of TCA cycle enzymes serves as a feedback mechanism down-regulating toxic respiration is discussed.

Analysis of protein carbonylation demonstrates that the stasis-induced catalases and cytoplasmic superoxide dismutases (SOD) have a role in preventing accelerated protein oxidation during growth arrest of Escherichia coli cells. A larger number of proteins are carbonylated in cells lacking cytoplasmic SOD compared with cells lacking catalases, OxyR, or RpoS which, in turn, exhibit a larger number of oxidized proteins than the wild-type parent. Proteins exclusively oxidized during stasis in mutants lacking cytoplasmic SOD include GroEL, EF-G, and the acidic isoform of H-NS indicating that these mutants experience problems in peptide elongation and maintaining protein and DNA architecture. These mutants also survive stasis poorly. Likewise, but to a much lesser extent, mutations in oxyR, an oxidative stress regulator, shorten the life-span of stationary phase cells. The low plating efficiency of cells lacking OxyR is the result of their inability to grow on standard culture plates unless plating is performed anaerobically or with high concentration of catalase. In contrast, cells lacking cytoplasmic SOD appear to die prior to plating. Our data points to the importance of oxidative stress defense in stasis survival, and we also demonstrate that the lifespan of growth-arrested wild-type E. coli cells can be significantly extended by omitting oxygen.Growth arrest of Escherichia coli cells caused by starvation for an essential nutrient triggers the production of catalases and other oxidative stress proteins which render the cell highly resistant to hydrogen peroxide, a phenomenon known as starvation-induced cross-protection (1-5). One model, we could call it the future provision model, suggests that this induction of stress defense proteins including oxidative stress proteins prepares the starved cell for stress conditions that it may encounter in the future. It has been argued that such a response is sensible at the onset of starvation because energy generation will become more and more limited as cells progress into stationary phase making inducible responses less immediate and less effective (6). Another model suggests that the induction of oxidative stress proteins has a role in minimizing damage to target molecules caused by stasis per se (3,7,8). Similarly, it has been proposed that the ubiquitous progressive decline in the functional capacity of aging eukaryotes is a consequence of the accumulation of oxidative damage caused by reactive oxygen species (ROS) 1 produced by normal metabolism (9). This is the postulation of the free radical hypothesis of aging (10). The hypothesis is supported by different experimental data demonstrating that (i) steady-state levels of oxidatively damaged macromolecules increase with age in all species examined thus far (11), (ii) oxidatively modified proteins lose their catalytic activity and structural integrity (12), (iii) there is a close association between oxidative damage of proteins and life expectancy of houseflies (13), and (iv) the life-span of fruitflies can be prolonge...

We have investigated the mechanisms of killing of Escherichia coli by HOCl by identifying protective functions. HOCl challenges were performed on cultures arrested in stationary phase and in exponential phase. Resistance to HOCl in both cases was largely mediated by genes involved in resistance to hydrogen peroxide (H 2 O 2 ). In stationary phase, a mutation in rpoS, which controls the expression of starvation genes including those which protect against oxidative stress, renders the cells hypersensitive to killing by HOCl. RpoSregulated genes responsible for this sensitivity were dps, which encodes a DNA-binding protein, and, to a lesser extent, katE and katG, encoding catalases; all three are involved in resistance to H 2 O 2 . In exponential phase, induction of the oxyR regulon, an adaptive response to H 2 O 2 , protected against HOCl exposure, and the oxyR2 constitutive mutant is more resistant than the wild-type strain. The genes involved in this oxyR-dependent resistance have not yet been identified, but they differ from those primarily involved in resistance to H 2 O 2 , including katG, ahp, and dps. Pretreatment with HOCl conferred resistance to H 2 O 2 in an OxyR-independent manner, suggesting a specific adaptive response to HOCl. fur mutants, which have an intracellular iron overload, were more sensitive to HOCl, supporting the generation of hydroxyl radicals upon HOCl exposure via a Fenton-type reaction. Mutations in recombinational repair genes (recA or recB) increased sensitivity to HOCl, indicative of DNA strand breaks. Sensitivity was visible in the wild type only at concentrations above 0.6 mg/liter, but it was observed at much lower concentrations in dps recA mutants.